Calcium-binding proteins, parvalbumin- and calbindin-D 28k-immunoreactive neurons in the rat spinal cord and dorsal root ganglia: a light and electron microscopic study

The distribution of two calcium-binding proteins, parvalbumin (PV) and calbindin-D 28K (CaBP), was studied by the peroxidase-anti-peroxidase immunohistochemical method at the light and electron microscopic level in the rat spinal cord and dorsal root ganglia. The possible coexistence of these two proteins was also investigated. PV-positive neurons were revealed in all layers of the spinal cord, except lamina I, which was devoid of labelling. Most of the PV-positive cells were found in the inner layer of lamina II, lamina III, internal basilar nucleus, central gray region, and at the dorsomedial and ventromedial aspects of the lateral motor column in the ventral horn. Neuronal processes intensely stained for PV sharply delineated inner lamina II. With the electron microscope most of them appeared to be dendrites, but vesicle containing profiles were also found in a smaller number. CaBP-positive neurons appeared to be dispersed all over the spinal gray matter. The great majority of them were found in laminae I, II, IV; the central gray region; the intermediolateral nucleus; and in the ventral horn just medial to the lateral motor column. Laminae I and II were densely packed with CaBP-positive punctate profiles that proved to be dendrites and axons in the electron microscope. A portion of labelled neurons in lamina IV and on the ventromedial aspect of the lateral motor column in the ventral horn disclosed both PV- and CaBP-immunoreactivity. All of the funiculi of the spinal white matter contained a large number of fibres immunopositive for both PV and CaBP. The highest density of CaBP-positive fibres was found in the dorsolateral funiculus, which was also densely packed with PV-positive fibres. PV-positive fibres were even more numerous in the dorsal part of the dorsal funiculus. The territory of the gracile funiculus in the brachial cord and that of the pyramidal tract in its whole extent were devoid of labelled fibres. In the thoracic cord, the dorsal nucleus of Clarke received a large number of PV-positive fibres. Dorsal root ganglia displayed both PV- and CaBP-immunopositivity. The cell diameter distribution histogram of PV-positive neurons disclosed two peaks--one at 35 microns and the other at 50 microns. CaBP-positive cells in the dorsal root ganglia corresponded to subgroups of small and large neurons with mean diameters of 25 microns and 45 microns, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical approach of GABAergic innervation of the mouse spinal cord using antibodies to GABA

The distribution of gamma-amino-butyric acid containing neurons in the Mouse spinal cord has been studied at both the light and electron microscope levels using antibodies against GABA and revelation by the Fab-peroxidase technique. At the light microscope level immunoreactive profiles of perikarya and neuronal processes were particularly abundant in the superficial laminae (I-IV) of the dorsal horn. Scattered soma profiles were found in the other layers and more particularly in the lamina X where Liquor contacting immuno-reactive neurons could be detected. GABAergic cell bodies were very sparse in the ventral horn. Electron microscopic observations confirmed the light microscope results: terminals constituted synaptic symmetrical contacts that provide a morphological basis for inhibition in the dorsal horn and for post-synaptic inhibition of motoneurons in the ventral horn.     

Development changes in the distribution of gamma-aminobutyric acid-immunoreactive neurons in the embryonic chick lumbosacral spinal cord

The development of γ-aminobutyric acid (GABA)-immunoreactive neurons was investigated in the embryonic and posthatch chick lumbosacral spinal cord by using pre- and postembedding immunostaining with an anti-GABA antiserum. The first GABA-immunoreactive cells were detected in the ventral one-half of the spinal cord dorsal to the lateral motor exception of the lateral motor column, appeared throughout the entire extent of the ventral one-half of the spinal gray matter by E6. Thereafter, GABA-immunoreactive neurons extended from ventral to dorsal regions. Stained perikarya first appeared at E8 and then progressively accumulated in the dorsal horn, while immunoreactive neurons gradually declined in the ventral horn. The general pattern of GABA immunoreactivity characteristic of mature animals had been achieved by E12 and was only slightly altered afterwards. In the dorsal horn, most of the stained neurons were observed in laminae I–III, both at the upper (LS 1–3) and at the lower (LS 5–7) segments of the lumbosacral spinal cord. In the ventral horn, the upper and lower lumbosacral segments showed marked differences in the distribution of stained perikarya. GABAergic neurons were scattered in a relatively large region dorsomedial to the lateral motor column at the level of the upper lumbosacral segments, whereas they were confined to the dorsalmost region of lamina VII at the lower segments. The early expression of GABA immunoreactivity may indicate a trophic and synaptogenetic role for GABA in early phases of spinal cord development. The localization of GABAergic neurons in the ventral horn and their distribution along the rostrocaudal axis of the lumbosacral spinal cord coincide well with previous physiological findings, suggesting that some of these GABAergic neurons may be involved in neural circuits underlying alternating rhythmic motor activity of the embryonic chick spinal cord. © 1994 Wiley-Liss, Inc.     

Neurogenesis in the mammalian neostriatum and nucleus accumbens: Parvalbumin-immunoreactive GABAergic interneurons

We study the neurogenesis of a distinct subclass of rat striatum γ-aminobutyric acid (GABA)ergic interneurons marked by the calcium-binding protein parvalbumin (PV). Timed pregnant rats are given an intraperitoneal injection of bromodeoxyuridine (BrdU), a marker of cell proliferation, on designated days between embryonic day (E) 11 and E22. Birthdate of PV neurons is determined in the adult neostriatum and nucleus accumbens by using a BrdU-PV double-labeling immunohistochemical technique. PV-immunoreactive interneurons of the neostriatum show maximum birthrates (>10% double-labeling) between E14–E17, whereas PV-immunoreactive interneurons of the nucleus accumbens show maximum double-labeling between E16–E19. In the neostriatum, caudal PV-immunoreactive neurons are born before those at rostral levels, and lateral PV-immunoreactive neurons become postmitotic before medial neurons. In the postcommissural striatum, ventral PV-immunoreactive neurons become postmitotic before dorsal neurons. In the precommissural striatum, ventral neurons are born before dorsal neurons laterally, but a dorsoventral gradient is seen medially. At corresponding coronal levels, PV-immunoreactive neurons of the nucleus accumbens are born shortly after PV neurons of the neostriatum. Analysis of BrdU labeling intensity in the nucleus accumbens shows that medium spiny projection neurons of the shell become postmitotic before neurons of the core. Similarly, PV-immunoreactive interneurons of the nucleus accumbens shell are born before PV interneurons of the core. Compared with cholinergic interneurons of the neostriatum, PV-immunoreactive interneurons are born later, but neurogenetic gradients are similar. The period of striatum PV interneuron genesis encompasses the period for somatostatin interneurons, although the latter neurons do not show neurogenetic gradients, possibly due to heterogeneous subtypes. Consideration of basal telencephalon neurogenesis suggests that subpopulations of striatum interneurons may share common neurogenetic features with phenotypically similar populations in the basal forebrain, with final morphology and connectivity depending on local cues provided by the host environment. J. Comp. Neurol. 389:193–211, 1997. © 1997 Wiley-Liss, Inc.     

Parvalbumin- and calbindin D28k-immunoreactive neurons in the superficial layers of the spinal cord dorsal horn of rat

Immunohistochemical techniques were utilized to investigate the distribution and morphology of neurons containing the calcium binding proteins parvalbumin (PV) and calbindin D28k (CaBP) in the superficial layers of rat spinal cord. Most PV-immunoreactive (PV-IR) neurons were restricted to a 25 to 60 microns thick band straddling the border between lamina II and III. Positive somata had long rostrocaudally oriented dendrites confined to narrow sagittally arranged sheets within this band and axons that entered lamina II or the superficial portions of lamina III. Long varicose axons, presumed to originate from these cells, were moderately distributed in Lissauer's tract and lamina II. CaBP-immunoreactive (CaBP-IR) neurons were found within lamina I and throughout lamina II. Large calibre PV-IR and CaBP-IR axons were seen in the dorsal column and the lateral funiculus. Dorsal rhizotomy or neonatal capsaicin treatment appeared to have no effect on PV-IR and CaBP-IR elements in the superficial lumbar dorsal horn. However, dorsal rhizotomy reduced the number of positive axons in the dorsal column and in deeper lamina of the dorsal horn. These results add to the known lamination patterns of the superficial dorsal horn and point to the existence of a lamina defined by PV-positive neurons at the lamina II/III border. These neurons may have electrophysiological characteristics attributed to PV- or CaBP-containing neurons elsewhere in the CNS.     

CalbindinD28k- and parvalbumin-immunoreactive neurons form complementary sublaminae in the rat superior colliculus

By using light microscopic immunocytochemistry and computer analysis, we have mapped the distributions of two calcium-binding proteins (CaBPs), calbindin_D28k (CB) and parvalbumin (PV), in the rat superior colliculus (SC). The patterns of CaBP expression were complementary. A band of heavily labeled, medium-sized CB-immunoreactive cells (CB-cells) was centered in the optic layer (OL), whereas PV-immunoreactive cells (PV-cells) were found predominantly in the intermediate gray layer (IGL), where they were clustered within patches of PV-labeled fibers. The superficial gray layer (SGL) could be divided into two sublaminae. CB-cells were found mostly in the dorsal half of the SGL, whereas PV-cells were scattered throughout the ventral SGL and the dorsal OL. Most of the CaBP-immunoreactive cells in the SGL were small bipolar cells with vertically oriented dendrites; however, there were also some PV-cells with horizontally oriented dendrites. Quantitative analysis of the CaBP distributions reinforced our observations that these cells are distributed in complementary tiers that are not restricted to the traditional laminae. The size and shape of some of these tiers were determined from a three-dimensional reconstruction of serial sections. The complementarity of the CaBP-immunoreactive tiers was also confirmed by fluorescence microscopy of double-labeled sections, in which few if any double-labeled neurons were observed. Complementary tiers of CB-cells and PV-cells have been observed previously in the SC of the cat. The present results demonstrate them in another species and further suggest that there are functional sublaminae in the SC that can be distinguished by CaBP content. J. Comp. Neurol. 394:205–217, 1998. © 1998 Wiley-Liss, Inc.     

Propriospinal afferent and efferent connections of the lateral and medial areas of the dorsal horn (laminae I-IV) in the rat lumbar spinal cord

The different subdivisions along the mediolateral extent of the superficial dorsal horn of the spinal cord are generally regarded as identical structures that execute the function of sensory information processing without any significant communication with other regions of the spinal gray matter. In contrast to this standing, here we endeavor to show that neural assemblies along the mediolateral extent of laminae I-IV cannot be regarded as identical structures. After injecting Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine into various areas of the superficial dorsal horn (laminae I-IV) at the level of the lumbar spinal cord in rats, we have demonstrated that the medial and lateral areas of the superficial dorsal horn show the following distinct features in their propriospinal afferent and efferent connections: 1) A 300- to 400-microm-long section of the medial aspects of laminae I-IV projects to and receives afferent fibers from a three segment long compartment of the spinal dorsal gray matter, whereas the same length of the lateral aspects of laminae I-IV projects to and receives afferent fibers from the entire rostrocaudal extent of the lumbar spinal cord. 2) The medial aspects of laminae I-IV project extensively to the lateral areas of the superficial dorsal horn. In contrast to this, the lateral areas of laminae I-IV, with the exception of a few fibers at the segmental level, do not project back to the medial territories. 3) There is a substantial direct commissural connection between the lateral aspects of laminae I-IV on the two sides of the lumbar spinal cord. The medial part of laminae I-IV, however, does not establish any direct connection with the gray matter on the opposite side. 4) The lateral aspects of laminae I-IV appear to be the primary source of fibers projecting to the ipsi- and contralateral ventral horns and supraspinal brain centers. Projecting fibers arise from the medial subdivision of laminae I-IV in a substantially lower number. The findings indicate that the medial and lateral areas of the superficial spinal dorsal horn of rats may play different roles in sensory information processing.     

Parvalbumin- and calbindin D28k-immunoreactive neurons in the hippocampal formation of the macaque monkey.

Calcium-binding proteins calbindin D28k (CaBP) and parvalbumin (PV) were localized in neurons of the monkey hippocampal formation. CaBP immunoreactivity is present in all granule cells and in a large proportion of CA1 and CA2 pyramidal neurons, as well as in a distinct population of local circuit neurons. In the dentate gyrus, CaBP-immunoreactive nongranule cells are present in the molecular layer and in the hilar region, but they do not include the pyramidal basket cells at the hilar border. In the Ammon's horn, CaBP-positive, nonpyramidal neurons are more frequent in the CA3 area than in any other parts of the hippocampal formation. They are concentrated in the strata oriens and pyramidale of areas CA1-3, whereas only a few small neurons were found in the strata lucidum and radiatum of CA3 and in the stratum moleculare of the CA1 area. PV is exclusively present in local circuit neurons both in the dentate gyrus and in Ammon's horn. In the dentate gyrus the presumed basket cells at the hilar border exhibit PV immunoreactivity. In the hilar region and molecular layer only a relatively small number of cells are immunoreactive for PV. Most of these PV-positive cell bodies are located in the inner half of the molecular layer, with occasional horizontal cells at the hippocampal fissure. In Ammon's horn, strata oriens and pyramidale of areas CA1-3 contain a large number of PV-positive cells. There are no PV-immunoreactive cells in the strata lucidum, radiatum, or lacunosum moleculare. The CaBP- and PV-containing neurons form different subpopulations of cells in the monkey hippocampal formation. With the exception of a basket cell type in the monkey dentate gyrus, the CaBP- and PV-positive cell types were found to be remarkably similar in rodents and primates.     

The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study.

The spinal course, termination pattern, and postsynaptic targets of the rubrospinal tract, which is known to contribute to the initiation and execution of movements, were studied in the rat at the light and electron microscopic levels by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with calbindin-D28k (CaBP), gamma-aminobutyric acid (GABA), and glycine immunocytochemistry. After injections of PHA-L unilaterally into the red nucleus, labelled fibers and terminals were detected at cervical, thoracic, and lumbar segments of the spinal cord. Most of the descending fibers were located in the dorsolateral funiculus contralateral to the injection site, but axons descending ipsilaterally were also revealed. Rubrospinal axon terminals were predominantly found in laminae V-VI and in the dorsal part of lamina VII at all levels and on both sides of the spinal cord, but stained collaterals were also seen in the ventrolateral aspect of Clark's column and in the ventral regions of lamina VII on both sides. The proportion of axonal varicosities revealed on the ipsilateral side varied at different segments and represented 10-28% of the total number of labelled boutons. Most of the labelled boutons were engaged in synaptic contacts with dendrites. Of the 137 rubrospinal boutons investigated, only 2 were found to establish axosomatic synaptic junctions in the lumbar spinal cord contralateral to the PHA-L injection. With the postembedding immunogold method, 80.8% of dendrites establishing synaptic contacts with rubrospinal terminals did not show immunoreactivity for either GABA or glycine, whereas 19.2% of them were immunoreactive for both amino acids. Rubrospinal axons made multiple contacts with CaBP-immunoreactive neurons in laminae V-VI. Synaptic contacts between rubrospinal terminals and CaBP-immunoreactive dendrites were identified at the electron microscopic level, and all CaBP-containing postsynaptic dendrites investigated were negative for both GABA and glycine. The results suggest that rubrospinal terminals establish synaptic contacts with both excitatory and inhibitory interneurons in the rat spinal cord, and a population of excitatory interneurons receiving monosynaptic rubrospinal input is located in laminae V-VI.     

A quantitative light and electron microscopic analysis of taurine-like immunoreactivity in the dorsal horn of the rat spinal cord.

Taurine has been proposed as an inhibitory neurotransmitter or neuromodulator in the vertebrate central nervous system. Within the spinal cord, taurine has been shown to have a direct inhibitory effect on spinal neurons and to have a selective antinociceptive effect on chemically induced nociception. Although sufficient data exists to suggest that taurine plays a neurotransmitter or neuromodulatory role in the spinal cord, it is not known whether this amino acid is present in axon terminals nor if this amino acid has a unique pattern of distribution within spinal tissue. To address these questions a monoclonal antibody against taurine was employed to localize taurine-like immunoreactivity in the dorsal horn of the rat spinal cord by using both light and electron microscopic techniques. Taurine-like immunoreactivity was most dense and most prominent in laminae I and II of the dorsal horn. A moderate amount of immunoreactivity was also present in laminae VIII and IX and X while the remaining laminae were only lightly stained. In laminae I and II taurine-like immunostaining was evident within neuronal cell bodies, dendrites, myelinated and unmyelinated axons, axon terminals, and astrocytes and their processes. Cell counts of these two laminae indicated that approximately 30% of neuronal perikarya at the C2 level, 52% of neuronal perikarya at the T6 level, and 18% of neuronal perikarya at the L2 level of the cord exhibited taurine-like immunoreactivity. With preembedding diaminobenzidine staining, approximately 20% of the axons examined in laminae I and II were found to be immunoreactive for taurine. Using postembedding immunogold staining in combination with quantitative procedures, the highest densities of gold particles were found in axon terminals containing pleomorphic vesicles and forming symmetrical synapses (36.8 particles/micron2), in a subpopulation of myelinated axons (34.2 particles/micron2), in a subpopulation of neuronal dendrites (32.6 particles/micron2), and in capillary endothelial cells (39.8 particles/micron2). Moderate labeling occurred in astrocytes (20.9 particles/micron2) and neuronal perikarya (18.7 particles/micron2). The localization of taurine to presumptive inhibitory axon terminals provides anatomical support for the hypothesis that taurine may serve an inhibitory neurotransmitter role in the superficial dorsal horn of the spinal cord. On the other hand, its localization to astrocytes and endothelial cells within both the dorsal ventral horns implies that it serves other nonneuronal functions as well.     

Immunohistochemical detection of calcium/calmodulin-dependent protein kinase II in the spinal cord of the rat and monkey with special reference to the corticospinal tract

Calcium/ calmodulin-dependent protein kinase II is a prominent enzyme in the mammalian brain that phosphorylates a variety of substrate proteins. In the present study, monoclonal antibodies that specifically recognize either the α or the β isoforms of this enzyme were used to determine the distribution of these isoforms within the rat and monkey spinal cord. In the rat, the corticospinal tract consists of two components: the dorsal corticospinal tract, which occupies the ventralmost aspect of the dorsal funiculus; and the ventral corticospinal tract, which occupies an area adjacent to the ventral median fissure. Both dorsal and ventral corticospinal tract fibers were strongly immunopositive for the α-antibody. Unilateral ablation of the sensorimotor cortex of the rat eliminated the α-immunoreactive staining in the contralateral dorsal corticospinal tract. The neuropil in the superficial laminae of the dorsal horn (Rexed's laminae I and II) was densely stained with the α-antibody, whereas the neuropil in laminae IV-X was immunonegative. Dense α-immunopositive neurons were also distributed in the head of the dorsal horn (laminae I-IV). In contrast to the strong α-immunoreactivity seen in the dorsal corticospinal tract fibers, only very weak β-immunoreactivity was observed in this tract. Moderate β-immunoreactive products were distributed homogenously throughout the neuropil of the gray matter, although the neuropil of the superficial laminae of the dorsal horn (laminae I and II) was stained more strongly than the other regions of the gray matter (laminae III-X). Neuronal components in all laminae were immunopositive for the β-antibody. Thus, motoneurons in the ventral horn, which were immunonegative for the α-antibody, were immunopositive for the β-antibody. This selective distribution pattern of immunoreactivity of α- and β-antibodies in the rat was also present in the monkey spinal cord, although the α-immunopositive corticospinal tract fibers in the monkey descended in the lateral funiculus as the lateral corticospinal tract instead of passing through the dorsal funiculus, as is the case in the rat. The differential distribution of immunoreactivity in the spinal cord suggests that these two isoforms of calcium/ calmodulin-dependent protein kinase II may have different functional roles in the spinal cord. © Wiley-Liss, Inc.     

Substance P- and GABA-like immunoreactivities are co-localized in axonal varicosities in the superficial laminae of cat but not rat spinal cord

In the present study, we applied a combination of pre-embedding peroxidase-based immunocytochemistry and post-embedding immunogold staining to examine the synaptic interactions of substance P (SP) and γ-aminobutyric acid (GABA) in the superficial laminae of the dorsal horn of cat and rat spinal cord. We demonstrate for the first time the co-existence of SP and GABA immunoreactivities in axonal boutons in laminae I–III of cat spinal dorsal horn. In cat, most SP + GABA immunoreactive (IR) axonal boutons stablished synapses with SP-IR or non-IR dendrites. These synapses were exclusively symmetric. Quantitative analysis showed that the percentage of SP/GABA double labelled bouton profiles was higher (7%) in lamina I but was considerably lower in laminae IIo, IIi and III. Similarly, the density (number of bouton profiles per 100 μm²) of SP + GABA-IR bouton profiles was highest in lamina I. However, in agreement with previous studies, the co-localization of SP and GABA immunoreactivities was never detected in the rat dorsal horn. In both species, SP + GABA-IR or GABA-IR axonal bouton profiles were never seen presynaptic to SP-IR boutons. These findings provide a morphological basis for the interaction of excitatory and inhibitory agents in the nociceptive circuits in the dorsal horn of the cat and rat spinal cord.     

Calcium-binding protein (calbindin-D28K) and parvalbumin immunocytochemistry in the normal and epileptic human hippocampus

The calcium-binding proteins calbindin-D28K (CaBP) and parvalbumin (PV) were localized in the "normal" and "epileptic" human hippocampus to address the possible relationship between the expression of these constitutive cytosolic calcium-binding proteins and the resistance or selective vulnerability of different hippocampal neuron populations in temporal lobe epilepsy. Compared to rodents and a baboon (Papio papio), the pattern of CaBP-like immunoreactivity (LI) in the "normal" human hippocampus is unique. CaBP-LI is present in the dentate granule cells, neurons of the "resistant zone" (area CA2), and presumed interneurons of all regions. Unlike rodent and baboon CA1 pyramidal cells, human CA1 pyramidal cells appear to be devoid of CaBP-LI. Thus, the relatively resistant dentate granule cells and CA2 pyramidal cells are the only human hippocampal principal cells that contain CaBP-LI normally. As in lower mammals, PV-LI is present exclusively in interneurons of all human hippocampal subregions. CaBP- and PV-LI were localized in hippocampi surgically removed in the treatment of intractable temporal lobe epilepsy to determine whether surviving hippocampal cells were those that express these calcium-binding proteins. Hippocampi removed from patients with tumors or arteriovenous malformations that were associated with complex partial seizures arising from this region appeared relatively normal histologically. CaBP- and PV-LI in this patient group appeared similar to that seen in autopsy controls. Conversely, "cryptogenic" epileptics, who exhibit hippocampal sclerosis as the only lesion associated with the epilepsy, exhibited a preferential survival of hippocampal cells that were CaBP- or PV-immunoreactive. In the dentate hilus, which normally contains few CaBP-LI neurons, most of the few surviving hilar neurons were CaBP-immunoreactive. Their number and darkness of staining suggests that CaBP synthesis may be increased in cells that survive. Despite an obvious decrease of PV-LI specifically in the damaged parts of the sclerotic hippocampi, PV-immunoreactive interneurons were often among the few surviving cells. Nevertheless, large expanses of the surviving granule cell layer appeared to have lost the PV-immunoreactive axosomatic fiber plexus. These results reveal a unique and striking correlation between the human hippocampal cells that normally express these calcium-binding proteins and those that survive in the sclerotic epileptic hippocampus.     

Patterns of expression of calcium binding proteins and neuronal nitric oxide synthase in different populations of hippocampal GABAergic neurons in mice

We examined the expression of calcium binding proteins parvalbumin (PV), calretinin (CR), and calbindin D28K (CB), and neuronal nitric oxide synthase (nNOS) in gamma-aminobutyric acid (GABA)ergic neurons of the mouse hippocampus, with particular reference to areal and dorsoventral differences. First, we estimated the colocalization of the calcium binding proteins and nNOS. GABAergic neurons containing both PV and nNOS, i.e., PV-immunoreactive (-IR)/nNOS-IR neurons, were rare in Ammon's horn but frequent in the dentate gyrus (DG). CR-IR/nNOS-IR neurons and CB-IR/nNOS-IR neurons were frequent in Ammon's horn but rare in the DG. In the entire hippocampus, the percentage of CR-IR neurons containing nNOS was significantly higher at the ventral level (44.3%) than at the dorsal level (17.0%). The percentage of CB-IR neurons containing nNOS was also significantly higher at the ventral level (42.3%) than at the dorsal level (29.3%). Next, we estimated the numerical densities (NDs) of calcium binding protein-containing GABAergic neurons. The ND of PV-IR neurons was comparable at the dorsal (1.16 x 10(3)/mm(3)) and ventral levels (1.23 x 10(3)/mm(3)), respectively. The ND of CR-IR neurons was less at the dorsal level (0.52 x 10(3)/mm(3)) than at the ventral level (0.64 x 10(3)/mm(3)). The ND of CB-IR neurons was also less at the dorsal level (0.91 x 10(3)/mm(3)) than at the ventral level (1.57 x 10(3)/mm(3)). Overall, approximately half of the GABAergic neurons contained one of the three calcium binding proteins (45% at the dorsal level and 47% at the ventral level). These data establish a baseline for examining potential roles of GABAergic neurons in hippocampal network activity in mice.     

Coexistence of NADPH diaphorase with GABA,glycine, and acetylcholine in rat spinal cord

The enzyme NADPH diaphorase is present in many spinal neurons, and is thought to correspond to nitric oxide synthase. In order to determine which types of neuron in the spinal cord contain this enzyme, we have carried out a combined enzyme histochemical and immunocytochemical study with antibodies to GABA, glycine, and choline acetyltransferase. Two hundred and twenty-four NADPH diaphorase-positive neurons in midlumbar spinal cord from four rats were tested for GABA- and glycine-like immunoreactivity. The majority of these neurons (207/224) were GABA-immunoreactive and 139 were also glycine-immunoreactive. NADPH diaphorase-positive neurons in laminae I and II generally showed both types of immunoreactivity, while those in deeper laminae of the dorsal horn and around the central canal either showed both types or else were only GABA-immunoreactive. Since GABA and acetylcholine are thought to coexist in spinal neurons, NADPH diaphorase staining was combined with immunostaining for choline acetyltransferase. Immunoreactive neurons in laminae III and IV were all NADPH diaphorase-positive, while only some of those around the central canal and in the deeper laminae of the dorsal horn were positive. Choline acetyltransferase-immunoreactive neurons in the intermediolateral cell column (presumed sympathetic preganglionic neurons) were often NADPH diaphorase-positive, whereas those in the ventral horn (presumed motorneurons) were not. NADPH diaphorase-positive cells in the intermediolateral cell column were not immunoreactive with GABA or glycine antibodies. These results suggest that NADPH diaphorase is largely restricted to GABAergic neurons in the lumbar spinal cord, and that it is mainly present in those neurons in which GABA coexists with glycine or acetylcholine. Since nitric oxide has been implicated in pain processing and hyperalgesia, while GABA, glycine, and acetylcholine are thought to be involved in analgesia and prevention of hyperalgesia, it is likely that nitric oxide synthase-containing GABAergic neurons in dorsal horn have dual actions in transmission of nociceptive information. © 1993 Wiley-Liss, Inc.     

Propriospinal pathways in the dorsal horn (laminae I-IV) of the rat lumbar spinal cord

The spinal dorsal horn is regarded as a unit that executes the function of sensory information processing without any significant communication with other regions of the spinal gray matter. Within the spinal dorsal horn, however, the different rostro-caudal and medio-lateral subdivisions intensively communicate with each other through propriospinal pathways. This review gives an overview about these propriospinal systems, and emphasizes that the medial and lateral parts of the spinal dorsal horn show the following distinct features in their propriospinal interconnectivities: (a) A 100-300μm long section of the medial aspects of laminae I-IV projects to and receives afferent fibers from a three segment long compartment of the spinal dorsal gray matter, whereas the same length of the lateral aspects of laminae I-IV projects to and receives afferent fibers from the entire rostro-caudal extent of the lumbar spinal cord. (b) The medial aspects of laminae I-IV project extensively to the lateral areas of the dorsal horn. In contrast to this, the lateral areas of laminae I-IV, with the exception of a few fibers at the segmental level, do not project back to the medial territories. (c) There is a substantial direct commissural connection between the lateral aspects of laminae I-IV on the two sides of the lumbar spinal cord. The medial part of laminae I-IV, however, establishes only a minor commissural propriospinal connection with the gray matter on the opposite side.     

Distribution of the tract of Lissauer and the dorsal root fibers in the primate spinal cord.

The tract of Lissauer receives small caliber dorsal root fibers in addition to axons arising from dorsal horn neurons. The termination of Lissauer's tract and dorsal root fibers was examined in the C7 segment of the rhesus monkey spinal cord. The distribution of normal dorsal root afferents was mapped by labelling the C7 dorsal root ganglion with tritiated amino acids, and then compared with the degeneration of C7 dorsal root fibers following an intradural dorsal rhizotomy. To focus on the distribution of the small afferents, the degeneration following a Lissauer tractotomy was compared with the degeneration following dorsal rhizotomy and following selected lesions involving the large afferents. The survival times following the lesions and rhizotomies were varied to facilitate identification of groups of fibers and terminals which might degenerate at different rates. Both large and small diameter dorsal root afferents were found to exhibit the same rostro-caudal topography within the dorsal horn. The C7 root axons and terminals distribute throughout the mid-C7 dorsal horn grey. Proceeding rostrally through C6, the majority of the C7 root fibers ending in laminae I-IV shift to a lateral position. Proceeding caudally through C8, the C7 root fibers shift medially. Few of the small diameter C7 afferents entering via Lissauer's tract extend above C6 or below C8. Large diameter C7 afferents, arising as dorsal column collaterals, can extend several segments above and below C7. Autoradiography revealed label in all dorsal horn laminae, the heaviest always occurring in the substantia gelatinosa. After one day, label was absent over dorsal column and Lissauer's tract axons, suggesting that the label was mainly associated with fine axonal branches or possibly terminals. After six to ten days many axons were labelled and could be traced into the dorsal and ventral horn. Degeneration from the rhizotomies and lesions, as demonstrated with Fink-Heimer and Nauta methods, depended on the survival time. No degeneration products were present before three days. The large afferents begin to degenerate within the dorsal horn after three to four days and mainly terminate in laminae IV-VI; by 12 days they can also be traced into the intermediate and ventral grey. The small afferents, which include those serving pain and temperature sensibility, arise from the tract of Lissauer and distribute to laminae I, II and III. The tract of Lissauer consists of two populations, each containing small afferents. One population degenerates at three to five days and distributes mainly to laminae II and III (substantia gelatinosa); the other degenerates around 12 days and distributes mainly to lamina I and the outer zone of II. It is suggested that the exclusive termination of the small afferents to laminae I, II and III may be correlated with certain unique histochemical properties (e.g., high substance P and high opiate receptor binding levels) of these same dorsal horn areas...     

GABA- and glycine-immunoreactive neurons in the spinal cord of the carp,Cyprinus carpio

γ-Aminobutyric acid (GABA) and glycine are the two main inhibitory transmitter amino acids in the central nervous system of vertebrates. The distribution of cells containing GABA and glycine in the carp spinal cord was examined by using specific antisera raised against the two amino acids conjugated to bovine serum albumin. The immunoreaction on serial paraffin sections was visualized by a streptavidin-biotin method. Both antisera gave highly specific labellings of cells. At least three types of GABA-immunoreactive cells were found. They were small cells in the dorsal grey matter, various sized cells in the central and ventral grey, and some ependymal cells contacting the central canal. In addition, very small cells and neuropil structures in the dorsal horn were strongly immunoreactive to the GABA serum. Certain cells in the ventral horn have moderate numbers of labelled synaptic boutons on the perikarya, but very few GABA-labelled terminals were found on putative motoneurons. The immunoreactive ependymal cells appeared to have a ventrolaterally directed axon. The glycine antiserum labelled small and intermediate cells in the dorsal grey, large, elongated cells in the median region, and varying sized cell sin the ventral grey. The numbers and density of immunoreactive cells and neuropil strucures in the ventral horn were fewer and lower than in GABA-stained sections. The median large cells had a thick venrolateral process. The ventral intermediate cells were often found near putative motoneurons. Labelled synaptic boutons were present on most ventral cells including putative motoneurons and interneurons. Abundant distribution of cells immunoreactive to both antisera suggest important roles of both GABA and glycine as neurotransmitters for controlling swimming movements in teleosts. © 1993 Wiley-Liss, Inc.     

Synaptic connections between trigemino-parabrachial projection neurons and γ-aminobutyric acid- and glycine-immunoreactive terminals in the rat

The synaptic connections between γ-aminobutyric acid (GABA)- and glycine-immunoreactive terminals and neurons projecting to the lateral parabrachial region were examined by a combination of retrograde tracing and immunohistochemical staining in the rat medullary dorsal horn. After injection of horseradish peroxidase (HRP) into the right lateral parabrachial region, HRP retrogradely labeled neurons were observed bilaterally in laminae I, II and III of the medullary dorsal horn with an ipsilateral predominance. GABA- and glycine-like immunoreactive terminals were found in laminae I, II and III. Some of these GABA- and glycine-like immunoreactive terminals were observed chiefly to make symmetric synapses with HRP-labeled neuronal cell bodies and dendritic processes. The present results indicate that neurons in the medullary dorsal horn projecting to the lateral parabrachial region might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons, which might be significantly involved in the regulation of the noxious information transmission.